Systematic Vertebrate Functional Genomics Abstract The vertebrate genome contains a predicted 20,000+ genes, many of unknown biological role(s). In addition, many of these molecules have distinct functions in diverse processes. The annotation of genes is one of the major next steps in understanding the vertebrate genome. We have developed an in vivo transposon trap of vertebrate secretory loci as a template for the generation of molecularly validated, morpholino-based knockdowns for members of the vertebrate secretome in the zebrafish as a part of a genome-wide assignment of protein function in a model vertebrate. Using this revised approach, we can continue to assign function to the 10-20% of the genome with morphologically visible phenotypes while new assays facilitate the assignment of function to the other 80% or more of the genome not discernible using standard genetic screening approaches. In this competitive renewal, we will focus on the systematic assignment of biological function to a set of 200+ secretory loci to identify new players in an array of clinically relevant developmental processes. We will accomplish this goal through the following specific aims: Aim I. Development of knockdown-verified morpholinos against 200+ secreted loci. Genes encoding in vivo validated secreted proteins will be identified through the use of a transposon-based secretory trap in living zebrafish. Morpholinos targeted to these genes will be generated and their knockdown verified before assignment of function in the following biological processes: Aim II. Isolation of secreted proteins required for organogenesis (including the pituitary and hypothalamus). Aim III. Isolation of secreted proteins required for cardiovascular formation including the choroid plexus and the behavioral genetics of the stress response. Aim IV. Isolation of secreted proteins required for digestive organ formation and lipid metabolism. Aim V. Annotation of the identified secretory transposon lines, functionally verified morpholinos, and phenotypes into the established, publicly available Morpholino Database (MODB), and international zebrafish databases at ZFIN and FishMap. The identification of molecules required for organogenesis, organ function and behavioral genetics such as the stress response has direct clinical implication. In addition, the molecules identified as crucial for development in vivo will serve as key substrate molecules for potential small molecule drug target intervention and for the establishment of conditions for in vitro stem cell manipulation. The zebrafish offers the first opportunity for a comprehensive analysis of these processes using as template an entire vertebrate genome.